Human T-cell lymphotropic virus type I (HTLV-I) encodes a 40 kD nuclear transactivating phosphoprotein, Tax1. Through interaction with cellular transcription factors, Tax1 regulates the level of viral and cellular gene expression. An in vitro transactivation assay, where wild type Tax1 is able to transactivate the HTLV-I promoter or a heterologous promoter that contains the Tax1 responsive elements, has been developed. The in vitro system utilizes the HTLV-I 21 base pair repeats cloned upstream of the ovalbumin promoter and G-free cassette. Purified Tax1 specifical ly transactivates this template five-to ten-fold in a concentration- dependent manner. Transactivation was effectively neutralized by anti-Tax1, but not control, sera. Transcription was inhibited by low concentrations of alpha-amanitin. No transactivation of the ovalbumin promoter template control was observed. Consistent with in vivo transactivating activity, Tax1 mutant M22 (CREB mutant) but not M47 (NF-kappaB mutant), transactivated the template containing the tandem 21 base pair repeat. In a reconstituted in vitro transcription assay, Tax1 transactivation was dependent upon basal transcription factors TFIID and TFIIA. TBP did not functionally substitute for TFIID in the transactivation assay. Tax1 transactivation of the HTLV-I long terminal repeat (LTR) has also been examined in vitro. In whole cell extracts, Tax1 transactivated the HTLV-I LTR but not the HIV LTR template. Both basal and Tax1-transactivated transcriptional activity of the HTLV-I LTR were resistant to low concentrations of alpha-amanitin. Human herpesvirus type 6 (HHV-6) is a lymphotropic virus isolated from peripheral blood lymphocytes (PBLs) of patients with lymphoproliferative disorders. HHV-6 DNA sequences have been identified in a number of human malignancies including African Burkitt's lymphoma and Epstein-Barr virus (EBV) negative B-cell lymphomas. HHV-6 may also be a cofactor in the progression of AIDS. It was demonstrated that the HHV-6 Sal1-L ORF-1 open reading frame encodes a 357 amino acid protein which transactivates the HIV LTR.